Deuterated glucose or fat tolerance tests for high-throughput measurement of the metabolism of sugars or fatty acids in the body

ABSTRACT

Provided herein are methods for determining the metabolism of one or more sugars and/or fatty acids, and applications thereof. Such applications include determining the rate of glycogen synthesis and glycolysis, which are believed to be early markers for predicting elevated risk of diabetes and cardiovascular disease. Other applications include methods for screening drugs that effect sugar and/or fatty acid metabolism. The methods are useful for at least partially characterizing drugs for desirable or undesirable (toxic) characteristics. Drugs that are at least partially characterized using the methods of the invention can then be further developed in pre-clinical testing and clinical trials. Such drugs may be found to be useful in treating obesity, diabetes, cardiovascular disease, and other disorders of metabolism.

RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional ApplicationSerial No. 60/423,964 filed Nov. 4, 2002, which is hereby incorporatedby reference in its entirety.

FIELD OF THE INVENTION

[0002] The present invention relates to the field of sugar and fattyacid metabolism. In particular, methods for determining the metabolismof one or more sugars or fatty acids in living organisms, includinghuman subjects, are described.

BACKGROUND OF THE INVENTION

[0003] Utilization of nutrients is key to many diseases, includingobesity, insulin resistance/diabetes mellitus, hyperlipidemia, andothers. The capacity to oxidize dietary fat relative to the tendency tostore ingested fat, for example, is considered to be a centraldeterminant of susceptibility to dietary fat-induced obesity. Similarly,the capacity to store or oxidize dietary glucose is a key element ininsulin resistance and glucose intolerance/diabetes. Tools for assessingthe fate of nutrients in the body in living organisms have laggedbehind, however. Currently available tools suffer from many limitations.

[0004] The oral glucose tolerance test (OGTT) is widely used in medicalresearch and clinical medicine for assessing insulin sensitivity oftissues. The principle of the OGTT is that uptake of glucose from bloodby tissues, along with suppression of release of endogenously producedglucose into blood from tissues, is reflected in the clearance rate ofan exogenous glucose load from the bloodstream. This approach is crude,however, and no information is generated about the specific metabolicfate or consequences of the glucose administered. As a result, noinformation is generated about the mechanisms underlying impairedglucose tolerance. Though widely used in clinical practice, the OGTT isof limited utility.

[0005] Fat tolerance testing has a similar basis and similar limitationsas OGTT. The fat tolerance test measures the uptake of fatty acids fromblood by tissues. This approach is also crude, and

[0006] Fat tolerance testing has a similar basis and similar limitationsas OGTT. The fat tolerance test measures the uptake of fatty acids fromblood by tissues. This approach is also crude, and gives no informationabout the specific metabolic fate or consequences of the fatadministered. As a result, no information is generated about themechanisms underlying impaired fat tolerance. Fat tolerance testing hasmostly been used to assess the clearance of dietary fat from blood incontext of evaluating hyperlipidemia. Fat tolerance testing is nothelpful for assessing sensitivity to high fat-induced obesity.

[0007] Indirect calorimetry (IC), or the measurement of fuel oxidationbased on respiration, is useful for whole body studies. IC, however, isexpensive and requires complex equipment for small animal studies. Also,IC only reveals the net oxidation of fuels in the whole body, withoutrevealing more details concerning the fate of individual fuels in thetissues.

[0008] Insulin/glucose clamps and other intensive approaches are oflimited practical utility in clinical practice or broad-based drugscreening/discovery, due to their labor-intensive nature. Physiologicrelevance is often also uncertain, since the procedures used (e.g.intravenous glucose infusion at high rates) do not mimic normalphysiologic intake of these nutrients.

[0009] The most direct approach is by use of isotopic techniques. Thesehave been highly problematic, however. The oxidation of ¹³C- or¹⁴C-labeled glucose or fatty acids to ¹³CO₂ or ¹⁴CO₂ has been used as amarker of tissue oxidation (1-3). The references cited herein are listedat the end of the specification before the claims. The serious flawswith this approach have been discussed previously (4). In brief,recovery of labeled CO₂ is a highly variable and unreliable index oftissue production of CO₂, due to re-utilization/exchange pathways of¹³CO₂ or ¹⁴CO₂. Yield of labeled CO₂ generated oxidatively in tissuescan be as low as 20%, or as high as 80% (1-4).

[0010] The most common risk factor setting for cardiovascular disease isthe so-called syndrome X or multiple risk factor syndrome (15) whereinan individual exhibits the combination of obesity, hypertension,hyperlipidemia, and glucose intolerance or diabetes. This syndrome isnow widely believed to be tied together pathogenically by insulinresistance, defined as lower-than-normal sensitivity of tissue to theeffects of insulin on glucose metabolism (15).

[0011] A primary component of tissue insulin resistance is impairment ofthe efficiency and rate of skeletal muscle and adipose tissue uptake andmetabolism of glucose in response to insulin exposure. One component oftissue glucose metabolism is storage as glycogen; the main alternativepathway for glucose metabolism in a tissue is glycolytic metabolism,leading to oxidation or other fates (FIGS. 2 and 3). Both the storage(non-oxidative) and glycolytic (oxidative) pathways are impaired ininsulin resistant tissues, such as skeletal muscle (15).

[0012] Because the insulin resistance syndrome is so common—indeed isthe most common medical abnormality in contemporary Westernpopulations—a reliable laboratory test for diagnosing and monitoringinsulin resistance has long been a very high priority. Variouscommentators have stated that a clinical marker of insulin resistancewould be a “holy grail in the fields of modern diabetes andcardiovascular disease” (C. Kahn, M. D., Director of ScientificSessions, American Diabetes Association, October 2003). The availabilityof a clinical test for insulin resistance would affect not only patientcare but also would allow drugs to be developed specifically to treatinsulin resistance.

[0013] Unfortunately, no current laboratory test is a reliable measureof insulin resistance. Serum insulin concentrations are highly variablefrom assay to assay and are influenced by insulin clearance as well astissue sensitivity to insulin. Other measures, such as bloodtriglyceride concentration, fasting glucose concentration, oral glucosetolerance, body mass index, waist-to-hip ratio, etc. correlate poorlywith clinical insulin sensitivity (as measured by a labor-intensiveresearch test, such as the insulin-glucose clamp technique; see Ref.15).

[0014] A technique for quantifying glucose metabolism by tissues—inparticular, glycolysis and/or glycogen storage of a glucose load—wouldtherefore have enormous impact on medical practice and drug trials.

SUMMARY OF THE INVENTION

[0015] In order to meet these needs, the present invention is directedto methods of determining the metabolism of one or more sugars or fattyacids, and uses of the methods in diagnosis and testing, and kits fordetermining the metabolism of one or more sugars and fatty acids.

[0016] In one format the invention disclosed herein represents areliable measure of insulin resistance, and reveals tissue insulinsensitivity or resistance in an individual. Use of the methods disclosedherein allows diagnostic classification of patients (for decisionsregarding risk-factor interventions), clinical monitoring of treatmentsintended to improve insulin sensitivity and reduce insulin resistance(such as the thiazolidinedones or metformin), and clinical developmentof new agents to treat insulin resistance (as an end-point or biomarkerof drug effect).

[0017] In one variation, the invention is directed to a method ofdetermining metabolism of one or more sugars or fatty acids in anindividual, where the method includes (a) administering one or morecompositions of one or more ²H-labeled sugars or ²H-labeled fatty acidsto an individual; (b) obtaining one or more bodily tissues or fluids atone or more times from the individual; and (c) detecting theincorporation of the ²H from the ²H-labeled sugars or ²H-labeled fattyacids into water to determine the sugar or fatty acid metabolism in theindividual.

[0018] In another variation, the one or more compositions include²H-labeled glucose. In another variation, the one or more compositionsinclude [6,6-²H₂]glucose, [1-²H₁]glucose, [3-²H₁]glucose,[2-²H₁]glucose, [5-²H₁]glucose, or [1,2,3,4,5,6-²H₇]glucose.

[0019] In another variation, the one or more compositions areadministered orally, by gavage, intraperitoneally, intravenously, orsubcutaneously. In a further variation, the one or more compounds areadministered orally.

[0020] In another variation, the individual is a mammal. In a furthervariation, the mammal is chosen from humans, rodents, primates,hamsters, guinea pigs, dogs, and pigs. In a still further variation, themammal is a human.

[0021] In another variation, the one or more bodily tissues or fluidsare chosen from blood, urine, saliva, and tears. In a further variation,the one or more bodily tissues or fluids are chosen from liver, muscle,adipose, intestine, brain, and pancreas.

[0022] In yet another variation, the water may be partially purified. Ina further variation, the water may be isolated.

[0023] In another variation, the method includes the additional step ofmeasuring ²H incorporation into one or more chemical compositions suchas glucose, glycogen, glycerol-triglyceride, triglyceride fatty acid,proteins, and DNA. In a further variation, the chemical composition isglucose. In a still further variation the method includes the additionalstep of measuring endogenous glucose production. In another variation,the method includes the additional step of measuring the proportion oflabeled glucose stored in tissue glycogen relative to sugaradministered. In yet another variation, the method includes theadditional step of measuring the proportion of administered ²H-glucoseundergoing glycolysis.

[0024] In another variation, the chemical composition is glycogen.

[0025] In another variation, the chemical composition isglycerol-triglyceride. In yet another variation, the method includes theadditional step of calculating new triglyceride synthesis.

[0026] In another variation, the chemical composition is triglyceridefatty acid. In still a further variation, the method includes theadditional step of calculating new fatty acid synthesis.

[0027] In another variation, the method includes the additional step ofcalculating the proportion of labeled fatty acids stored in tissuerelative to labeled fatty acid administered. In a further variation, themethod includes the additional step of calculating the proportion ofadministered labeled fatty acids undergoing fatty acid oxidation.

[0028] In another variation, the chemical composition is a protein.

[0029] In yet another variation, the chemical composition is DNA. In afurther variation, the method includes the additional step ofcalculating the rate of DNA synthesis.

[0030] In another variation, the method of determining sugar or fattyacid metabolism in an individual further includes calculating the rateof incorporation of ²H into the water. In another variation, the methodincludes calculating the rate of incorporation of ²H into one or morechemical compositions such as glucose, glycogen, glycerol-triglyceride,triglyceride fatty acid, proteins, and DNA. Optionally, both the ratesof water formation and chemical composition formation may be monitored.

[0031] In another variation, the water may be detected by gaschromatography/mass spectrometry, liquid chromatography-massspectrometry, gas chromatography-pyrolysis-isotope ratio/massspectrometry, or gas chromatography-combustion-isotope ratio/massspectrometry, cycloidal mass spectrometry, Fourier-transform-isotoperatio (IR)-spectroscopy, near IR laser spectroscopy, or isotope ratiomass spectrometry.

[0032] In a still further variation, the detecting step may beaccomplished by detecting one part ²H in 10⁷ parts water.

[0033] In another aspect, the invention also includes furtherapplications of the methods of the invention to determine the metabolismof sugars and fatty acids. In one variation, a drug agent is introducedto the individual prior to determining the metabolism of one or moresugars or fatty acids, and the effect on an individual is subsequentlyidentified. In another variation, the metabolism determinations are usedas a surrogate marker for FDA or other regulatory agency approval ofdrugs. In yet another variation, the metabolism determination is usedfor the clinical management of patients. In still a further variation,the metabolism determination includes diagnosing, prognosing, oridentifying individuals at risk for insulin resistance/diabetes mellitusin the individual. In another variation, the metabolism determinationincludes diagnosing, or identifying individuals at risk for, high-fatdiet-induced obesity. In still another variation, the metabolismdetermination includes monitoring the effects of interventions toprevent or reverse insulin resistance/diabetes mellitus or high-fatdiet-induced obesity. In another variation, the metabolism determinationincludes diagnosing or treating wasting disorders, hypoglycemia, orglycogen storage disease.

[0034] The invention is also directed to drug agents that are identifiedas having an effect on the sugar or fatty acid metabolism of theindividual, and isotopically perturbed molecules such as glucose,glycogen, glycerol-triglyceride, triglyceride fatty acid, proteins, andDNA.

[0035] The invention is further directed to a kit for determining themetabolism of a sugar in an individual. The kit may include one or morelabeled sugars and instructions for use of the kit. The kit is usefulfor determining sugar metabolism in an individual. The kit may furtherinclude chemical compounds for isolating water. The kit may also includechemical compounds for isolating glucose, glycogen, proteins or DNA. Thekit may also include a tool for administering labeled glucose. The kitmay further include an instrument for collecting a sample from theindividual.

[0036] The invention is further directed to a drug agent the effect ofwhich was at least partially identified by the methods of the invention.

[0037] The invention is further directed to an isotopically perturbedmolecule chosen from glycogen, glycerol-triglyceride, triglyceride fattyacid, proteins, and DNA.

[0038] The invention is further directed to a method of manufacturingone or more drug agents at least partially identified by the methods ofthe invention.

[0039] The invention is further directed to an information storagedevice including data obtained from the methods of the invention. Thedevice may be a printed report or a computer. The printed report may beprinted on paper, plastic, or microfiche. The device may be a computerdisc. The computer disk may be chosen from a compact disc, a digitalvideo disc, and a magnetic disc.

BRIEF DESCRIPTION OF THE DRAWINGS

[0040]FIG. 1 depicts the fate of ²H attached to fatty acids in thecells. In this case, palmitate is shown. The fatty acid is metabolizedvia β-oxidation to release hydrogen atoms from C—H bonds of fats to bodyH₂O. Alternatively, fatty acids may be esterified to producetriglyceride-fatty acids, in this case triglyceride-palmitate.

[0041]FIG. 2 depicts the fate of ²H attached to sugars, in this caseglucose. Sugars are metabolized via glycolysis and the citric acid cycleto release hydrogen atoms from C—H bonds of sugars to body H₂O.Alternatively, glucose may form glycogen.

[0042]FIG. 3 depicts a schematic molecule of glucose or fat tolerancetests. Glucose or fatty acid metabolism may be measured directly fromrelease of ²H to body water. Measurements may include the additionalstep of incorporating ²H from body water back into other labeledchemical compounds, including labeled glycerol-triglycerides, fattyacid-triglycerides, proteins, DNA, or components thereof.

[0043]FIG. 4 depicts a kinetic oral glucose tolerance test in a normalhuman subject. The percent glycolysis, measured by deuteriumincorporation into water following administration of deuterium-labeledglucose, is shown over a period of time.

[0044]FIG. 5 depicts a kinetic oral glucose tolerance test in a normalmouse. The percent glycolysis, measured by deuterium incorporation intowater following administration of deuterium-labeled glucose, is shownover a period of time.

[0045]FIG. 6 depicts a kinetic oral glucose tolerance test in a normalrat. The percent glycolysis, measured by deuterium incorporation intowater following administration of deuterium-labeled glucose, is shownover a period of time.

DETAILED DESCRIPTION OF THE INVENTION

[0046] A method for determining the metabolism of ²H-labeled sugars andfatty acids is described herein. The methods have numerous applicationsin the fields of medical diagnostics and biological research.

I. General Techniques

[0047] The practice of the present invention will employ, unlessotherwise indicated, conventional techniques of molecular biology(including recombinant techniques), microbiology, cell biology,biochemistry, immunology, protein kinetics, and mass spectroscopy, whichare within the skill of the art. Such techniques are explained fully inthe literature, such as, Molecular Cloning: A Laboratory Manual, secondedition (Sambrook et al., 1989) Cold Spring Harbor Press;Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methods in MolecularBiology, Humana Press; Cell Biology: A Laboratory Notebook (J. E.Cellis, ed., 1998) Academic Press; Animal Cell Culture (R. I. Freshney,ed., 1987); Introduction to Cell and Tissue Culture (J. P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: LaboratoryProcedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds., 1993-8)J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.);Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell,eds.); Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P.Calos, eds., 1987); Current Protocols in Molecular Biology (F. M.Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mulliset al., eds., 1994); Current Protocols in Immunology (J. E. Coligan etal., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons,1999); and Mass isotopomer distribution analysis at eight years:theoretical, analytic and experimental considerations by Hellerstein andNeese (Am J Physiol 276 (Endocrinol Metab. 39) E1146-E1162, 1999).Furthermore, procedures employing commercially available assay kits andreagents will typically be used according to manufacturer-definedprotocols unless otherwise noted.

II. Definitions

[0048] Unless otherwise defined, all terms of art, notations and otherscientific terminology used herein are intended to have the meaningscommonly understood by those of skill in the art to which this inventionpertains. In some cases, terms with commonly understood meanings aredefined herein for clarity and/or for ready reference, and the inclusionof such definitions herein should not necessarily be construed torepresent a substantial difference over what is generally understood inthe art. The techniques and procedures described or referenced hereinare generally well understood and commonly employed using conventionalmethodology by those skilled in the art, such as, for example, Massisotopomer distribution analysis at eight years: theoretical, analyticand experimental considerations by Hellerstein and Neese (Am J Physiol276 (Endocrinol Metab. 39) E1146-E1162, 1999), herein incorporated byreference. As appropriate, procedures involving the use of commerciallyavailable kits and reagents are generally carried out in accordance withmanufacturer defined protocols and/or parameters unless otherwise noted.

[0049] “Metabolism” is used interchangeably with “metabolic fate” and“metabolic consequences,” and refers generally to biosynthesis,breakdown, conversion, oxidation, and/or reduction of sugars and fattyacids.

[0050] “Isotopes” refer to atoms with the same number of protons andhence of the same element but with different numbers of neutrons (e.g.,Hydrogen (H) vs. Deuterium (D)). D is also represented as ²H, as iscommon in the art.

[0051] “Isotopomers” refer to isotopic isomers or species that haveidentical elemental compositions but are constitutionally and/orstereochemically isomeric because of isotopic substitution, as forCH₃NH₂, CH₃NHD and CH₂DNH₂.

[0052] “Isotopologues” refer to isotopic homologues or molecular speciesthat have identical elemental and chemical compositions but differ inisotopic content (e.g., CH₃NH₂ vs. CH₃NHD in the example above).Isotopologues are defined by their isotopic composition, therefore eachisotopologue has a unique exact mass but may not have a uniquestructure. An isotopologue is usually included of a family of isotopicisomers (isotopomers) which differ by the location of the isotopes onthe molecule (e.g., CH₃NHD and CH₂DNH₂ are the same isotopologue but aredifferent isotopomers).

[0053] “Mass isotopomer” refers to a family of isotopic isomers that aregrouped on the basis of nominal mass rather than isotopic composition. Amass isotopomer may comprise molecules of different isotopiccompositions, unlike an isotopologue (e.g., CH₃NHD, ¹³CH₃NH₂, CH₃ ¹⁵NH₂are part of the same mass isotopomer but are different isotopologues).In operational terms, a mass isotopomer is a family of isotopologuesthat are not resolved by a mass spectrometer. For quadrupole massspectrometers, this typically means that mass isotopomers are familiesof isotopologues that share a nominal mass. Thus, the isotopologuesCH₃NH₂ and CH₃NHD differ in nominal mass and are distinguished as beingdifferent mass isotopomers, but the isotopologues CH₃NHD, CH₂DNH₂,¹³CH₃NH₂, and CH₃ ¹⁵NH₂ are all of the same nominal mass and hence arethe same mass isotopomers. Each mass isotopomer is therefore typicallycomposed of more than one isotopologue and has more than one exact mass.The distinction between isotopologues and mass isotopomers is useful inpractice because all individual isotopologues are not resolved usingquadrupole mass spectrometers and may not be resolved even using massspectrometers that produce higher mass resolution, so that calculationsfrom mass spectrometric data must be performed on the abundances of massisotopomers rather than isotopologues. The mass isotopomer lowest inmass is represented as M₀; for most organic molecules, this is thespecies containing all ¹²C, ¹H, ¹⁶O, ¹⁴N, etc. Other mass isotopomersare distinguished by their mass differences from M₀ (M₁, M₂, etc.). Fora given mass isotopomer, the location or position of isotopes within themolecule is not specified and may vary (i.e., “positional isotopomers”are not distinguished).

[0054] “Mass isotopomer pattern” refers to a histogram of the abundancesof the mass isotopomers of a molecule. In one embodiment, the pattern ispresented as percent relative abundances where all of the abundances arenormalized to that of the most abundant mass isotopomer; the mostabundant isotopomer is said to be 100%. In another embodiment, the formfor applications involving probability analysis is proportion orfractional abundance, where the fraction that each species contributesto the total abundance is used (see below). The term isotope pattern issometimes used in place of mass isotopomer pattern, although technicallythe former term applies only to the abundance pattern of isotopes in anelement.

[0055] An “individual” refers to a vertebrate animal including a mammaland further including a human.

[0056] A “biological sample” encompasses a variety of sample typesobtained from an individual. The definition encompasses blood and otherliquid samples of biological origin, that are accessible from anindividual through sampling by minimally invasive or non-invasiveapproaches (e.g., urine collection, blood drawing, needle aspiration,and other procedures involving minimal risk, discomfort or effort). Thedefinition also includes samples that have been manipulated in any wayafter their procurement, such as by treatment with reagents,solubilization, or enrichment for certain components, such as proteinsor polynucleotides. The term “biological sample” also encompasses aclinical sample such as serum, plasma, other biological fluid, or tissuesamples, and also includes cells in culture, cell supernatants and celllysates.

[0057] “Biological fluid” includes but is not limited to urine, blood,blood serum, amniotic fluid, interstitial fluid, edema fluid, saliva,lacrimal fluid, inflammatory exudates, synovial fluid, abscess, empyemaor other infected fluid, cerebrospinal fluid, sweat, pulmonarysecretions (sputum), seminal fluid, feces, bile, intestinal secretions,conjunctival fluid, tears, vaginal fluid, stool, or other bodily fluid.

[0058] “Sugar” refers to a monosaccharide or a polysaccharide comprisedof monosaccharide residues. Examples of monosaccharides include, but arenot limited to, glucose (both D-glucose and L-glucose), mannose,fructose galactose and sugar derivatives such as glucoronic acid,glucosamine. Examples of polysaccharides include, but are not limitedto, disaccharides such as sucrose, maltose and lactose and longer chainsugar molecules such as glycogen.

[0059] “Labeled sugar” refers to a sugar incorporating one or more ²Hisotopes.

[0060] “Labeled fatty acid” refers to a fatty acid incorporating one ormore ²H isotopes. “Deuterated water” refers to water incorporating oneor more ²H isotopes. “Labeled glucose” refers to glucose labeled withone or more ²H isotopes. Specific examples of labeled glucose or²H-labeled glucose include [6,6-²H₂]glucose, [1-²H₁]glucose, and[1,2,3,4,5,6-²H₇]glucose.

[0061] “Partially purifying” refers to methods of removing one or morecomponents of a mixture of other compounds. For example, “partiallypurifying one or more proteins or peptides” refers to removing one ormore proteins or peptides from a mixture of one or more proteins orpeptides or other compounds. As another example, “partially purifyingwater” refers to removing one or more molecules, such as macromolecules,types of macromolecules, or salts, from water.

[0062] “Isolating” refers to separating one compound from a mixture ofcompounds. For example, “isolating one or more proteins or peptides”refers to separating one protein or peptide from a mixture of one ormore proteins or peptides or other compounds. “Isolating water” refersto removing all additional compounds beyond trace levels from water.

[0063] “Drug agent,” “pharmaceutical agent,” “pharmacological agent,”and “pharmaceutical” are used interchangeably to refer to any chemicalentities, known drug or therapy, approved drug or therapy, biologicalagent (e.g., gene sequences, poly or monoclonal antibodies, cytokines,and hormones). Drug agents include, but are not limited to, any chemicalcompound or composition disclosed in, for example, the 13th Edition ofThe Merck Index (a U.S. publication, Whitehouse Station, N.J., USA),incorporated herein by reference in its entirety.

[0064] “Isotopically perturbed” refers to the state of an element ormolecule that results from the explicit incorporation of an element ormolecule with a distribution of isotopes that differs from thedistribution that is most commonly found in nature, whether a naturallyless abundant isotope is present in excess (enriched) or in deficit(depleted).

[0065] “At least partially identified” in the context of drug discoveryand development means at least one clinically relevant pharmacologicalcharacteristic of a drug agent has been identified using one or more ofthe methods of the present invention. This characteristic may be adesirable one, for example, increasing or decreasing molecular fluxrates through a metabolic pathway that contributes to a disease process,altering signal transduction pathways or cell surface receptors thatalter the activity of metabolic pathways relevant to a disease,inhibiting activation of an enzyme and the like. Alternatively, apharmacological characteristic of a drug agent may be an undesirable onefor example, the production of one or more toxic effects. There are aplethora of desirable and undesirable characteristics of drug agentswell known to those skilled in the art and each will be viewed in thecontext of the particular drug agent being developed and the targeteddisease. Of course, a drug agent can be more than at least partiallyidentified when, for example, when several characteristics have beenidentified (desirable or undesirable or both) that are sufficient tosupport a particular milestone decision point along the drug developmentpathway. Such milestones include, but are not limited to, pre-clinicaldecisions for in vitro to in vivo transition, pre-IND filing go/no godecision, phase I to phase II transition, phase II to phase IIItransition, NDA filing, and FDA approval for marketing. Therefore, “atleast partially” identified includes the identification of one or morepharmacological characteristics useful in evaluating a drug agent in thedrug discovery/drug development process. A pharmacologist or physicianor other researcher may evaluate all or a portion of the identifieddesirable and undesirable characteristics of a drug agent to establishits therapeutic index. This may be accomplished using procedures wellknown in the art.

[0066] “Manufacturing drug agents” in the context of the presentinvention includes any means, well known to those skilled in the art,employed for the making of a drug agent product. Manufacturing processesinclude, but are not limited to, medicinal chemical synthesis (i.e.,synthetic organic chemistry), combinatorial chemistry, biotechnologymethods such as hybridoma monoclonal antibody production, recombinantDNA technology, and other techniques well known to the skilled artisan.Such a product may be a final drug agent that is marketed fortherapeutic use, a component of a combination product that is marketedfor therapeutic use, or any intermediate product used in the developmentof the final drug agent product, whether as part of a combinationproduct or a single product.

[0067] “Elevated risk” is used interchangeably herein with “increasedrisk” and means an increase, beyond measurable background levels, of therisk of an individual for acquiring a condition or disease based on thepresence or absence of one or more risk factors.

[0068] “Risk factor” as used herein, means an external or internalfactor that is associated with a disease or disorder. A risk factor mayreflect an aspect of causation, whether direct or indirect, but is notso limited. A risk factor may have an association with the onset of adisease or disorder and may be predictive of such (i.e., a marker ofdisease), but may or may not be an indicator of the underlying pathologyof the disease or disorder.

III. Methods of the Invention

[0069] Non-invasive tests for determining the metabolism of metabolitessuch as sugars and fatty acids in the body have great utility forclinical diagnostics and biomedical research. We disclose here methodsthat allow high-throughput, inexpensive and simple measurements of thedisposal pathways and metabolic consequences of sugars and fatty acidsin living organisms, including humans. As used herein “metabolism,”“metabolic fate” and “metabolic consequences” are used interchangeablyand refer generally to biosynthesis, breakdown, conversion, oxidation,and/or reduction of sugars or fatty acids upon administration. The testinvolves determining the metabolism of sugars and fatty acids byadministering one or more isotope labeled sugars or labeled fatty acidsto an individual, then detecting the release of the label in bodilytissues or fluids to determine the metabolism of the one or more sugarsor fatty acids in the individual. In one embodiment, the test involvesadministration of deuterium-labeled glucose or fatty acids to a humansubject or experimental animal, then measurement of the release ofdeuterium to body water. Highly sensitive measurements of labelenrichments in chemical compositions contained in the bodily tissues orfluids allow great sensitivity and accuracy by this approach.

[0070] The tests disclosed herein have utility as drug discovery tools(e.g., for identifying genes and drugs that alter tissue glucose or fatutilization pathways); as surrogate biomarkers for FDA approval of drugs(e.g., agents influencing fat oxidation or insulin sensitivity oftissues); and as diagnostic measures for the clinical management ofpatients. The methods may be used to diagnose, or identify, the risk ofinsulin resistance or diabetes mellitus. The methods may also be used toidentify diet-induced obesity or the risk of acquiring diet-inducedobesity. The methods may further be used to diagnose or treat wastingdiseases and disorders. Further, the methods may also be used toidentify hypoglycemia or hyperglycemia. In addition, the methods may beused to diagnose or treat glycogen storage diseases. By measuring thetotal disappearance of glucose (D_(glucose)) and the formation ofglycolysis (as described, infra), the rate of glycogen synthesis and/orthe concentration (i.e., the amount) of glycogen synthesized (i.e.,formed) can then be determined. Knowing the rate of glycogen synthesisand/or the amount of glycogen formed, for example, enables the clinicianto evaluate the efficacy of drug agents intended to improve tissueinsulin sensitivity (e.g., in pre-diabetic individuals) or in treatingglycogen storage diseases. Alternatively, knowing the rate of glycogensynthesis and/or the amount of glycogen formed allows the clinician tomore accurately diagnose or prognose a glycogen storage disease.Additionally, the rate of glycogen synthesis and/or the amount ofglycogen formed is a well-accepted early marker for an elevated risk ofdeveloping cardiovascular disease or insulin-resistant disorders such astype II diabetes.

[0071] The invention disclosed herein combines the simplicity of an OGTTor fat tolerance test with the precision, accuracy and metabolicspecificity of deuterium tracing. Partitioning labeled ²H, attached tospecific C—H bonds of administered compounds such as sugars or fattyacids, can reveal the specific metabolic fate of the nutrient in aliving organism and can be monitored in a high-throughput, inexpensivemanner.

Methods of Determining the Metabolism of Compositions Containing Sugarsor Fatty Acids in an Individual

[0072] i) Administering Labeled Metabolites to an Individual

[0073] a. Compositions Containing Sugars

[0074] Compositions containing sugars may include monosaccharides,polysaccharides, or other compounds that are covalently bonded tomonosaccharides or polysaccharides.

[0075]²H-labeled sugars may be administered to an individual asmonosaccharides or as polymers of monosaccharide residues. Labeledmonosaccharides may be readily obtained commercially (for example,Cambridge Isotopes, Massachusetts).

[0076] Relatively low quantities of compositions that contain ²H-labeledsugars need to be administered. Quantities may be on the order ofmilligrams, 10¹ mg, 10² mg, 10³ mg 10⁴ mg, 10⁵ mg, or 10⁶ mg. ²H-labeledsugar enrichment may be maintained for weeks or months in humans and inanimals without any evidence of toxicity. The lower cost of commerciallyavailable labeled monosaccharides, and low quantity that need to beadministered, allow maintenance of enrichments at low expense.

[0077] In one particular variation, the labeled sugar is glucose. FIG. 2shows the fate of ²H-labeled glucose. Glucose is metabolized byglycolysis and the citric acid cycle. Glycolysis releases most of theH-atoms from C—H bonds of glucose; oxidation via the citric acid cycleensures that all H-atoms are released to H₂O. In a further variation,the labeled glucose may be [6,6-²H₂]glucose, [1-²H]glucose, and[1,2,3,4,5,6-²H₇]glucose.

[0078] In another variation, labeled sugar may be fructose or galactose.Fructose is metabolized via the fructose 1-phosphate pathway, andsecondarily through phosphorylation to fructose 6-phosphate byhexokinase. Galactose is metabolized via the galactose to glucoseinterconversion pathway.

[0079] Any other sugar may be utilized in the disclosed methods. Othermonosaccharides, include, but are not limited to, trioses, pentoses,hexose, and higher order monosaccharides. Monosaccharides furtherinclude, but are not limited to, aldoses and ketoses.

[0080] In another variation, compositions containing polysaccharides maybe administered. The polymers may be formed from monosaccharides: Forexample, labeled glycogen, a polysaccharide, is formed by glucoseresidues. In another variation, labeled polysaccharides may beadministered. As further variation, labeled sugar monomers may beadministered as a component of sucrose (glucose α-(1,2)-fructose),lactose (galactose β-(1,4)-glucose), maltose (glucose α-(1,4)-glucose),starch (glucose polymer), or other polymers.

[0081] In another variation, the labeled sugar may be administeredorally, by gavage, intraperitoneally, intravascularly includingintra-arterially and intravenously, subcutaneously, or other bodilyroutes. In particular, the sugars may be administered to an individualorally, optionally as part of a food or drink. By “administering” or“administration” is meant any method that introduces the labeled sugarto, in or on an individual.

[0082] The individual may be a mammal. In one embodiment, the individualmay be an experimental mammal. In another embodiment, the individual maybe a rodent, primate, hamster, guinea pig, dog, or pig. In yet anotherembodiment, the individual may be a human.

[0083] b. Labeled Fatty Acids

[0084] Determining the metabolism of compounds that contain ²H-labeledfatty acids is also included in this invention.

[0085]²H-labeled fatty acids may be administered to an individual asfats or other compounds containing the labeled fatty acids. ²H-labeledfatty acids may be readily obtained commercially. Relatively lowquantities of labeled fatty acids need to be administered. Quantitiesmay be on the order of milligrams, 10¹ mg, 10² mg, 10³ mg, 10⁴ mg, 10⁵mg, or 10⁶ mg. Fatty acid enrichment, particularly with ²H, may bemaintained for weeks or months in humans and in animals without anyevidence of toxicity. The lower cost of commercially available labeledfatty acids, and low quantity that need to be administered, allowmaintenance of enrichments at low expense.

[0086]FIG. 1 shows the fate of ²H-labeled fatty acids during β-oxidation(metabolism) of fatty acids in cells. β-oxidation releases hydrogenatoms from C—H bonds of fats to body H₂O. All H-atoms are released from²H-fatty acids during β-oxidation and, once β-oxidation starts on afatty acid, the process goes to completion. The release of labeled fattyacids, particularly ²H-fatty acid, to labeled water, particularly ²H₂O,accurately reflects fat oxidation. Administration of modest amounts oflabeled-fatty acid is sufficient to measure release of labeled hydrogenor oxygen to water. In particular, administration of modest amounts of²H-fatty acid is sufficient to measure release of ²H to deuterated water(i.e., ²H₂O).

[0087] Relatively low quantities of labeled fatty acid or fatty acidresidue need to be administered. Quantities may be on the order ofmilligrams, 10¹ mg, 10² mg, 10³ mg, 10⁴ mg, 10⁵ mg, or 10⁶ mg.²H-labeled fatty acid enrichment may be maintained for weeks or monthsin humans and in animals without any evidence of toxicity. The lowerexpense of commercially available labeled fatty acids and fatty acidresidues, and low quantity that need to be administered, allowmaintenance of enrichments at low expense.

[0088] In another variation, the labeled fatty acids may be administeredorally, by gavage, intraperitoneally, intravascularly includingintra-arterially and intravenously, subcutaneously, or other bodilyroutes. In particular, the labeled fatty acids may be administered to anindividual orally, optionally as part of a food or drink. By“administering” or “administration” is meant any method that introducesthe labeled fatty acid to, in or on an individual.

[0089] The individual may be a mammal. In one embodiment, the individualmay be an experimental mammal. In another embodiment, the individual maybe a rodent, primate, hamster, guinea pig, dog, or pig. In yet anotherembodiment, the individual may be a human.

[0090] (ii) Obtaining One or More Bodily Tissues or Fluids from SaidIndividual

[0091] A biological sample is obtained from bodily tissues or fluids ofan individual. Specific methods of obtaining biological samples are wellknown in the art. Bodily fluids include, but are not limited to, urine,blood, blood serum, amniotic fluid, spinal fluid, conjunctival fluid,saliva, tears, vaginal fluid, stool, seminal fluid, and sweat. Thefluids may be isolated by standard medical procedures known in the art.Bodily tissues include, but are not limited to, liver, muscle, adipose,intestine, brain, and pancreas.

[0092] In one variation, water may be partially purified. In anothervariation, the water may be isolated.

[0093] In another variation, the one or more bodily tissue or fluids maybe obtained after a period of time. In a further variation, the one ormore bodily tissues or fluids may be obtained multiple times.

[0094] iii) Detecting the Incorporation of ²H Into Water

[0095] a. Mass Spectrometry

[0096] The isotope label, or alternatively, the labeled chemicalcompositions, may be determined by various methods such as massspectrometry, particularly gas chromatography-mass spectrometry (GC-MS).Incorporation of labeled isotopes into chemical compositions may bemeasured directly. Alternatively, incorporation of labeled isotopes maybe determined by measuring the incorporation of labeled isotopes intoone or more hydrolysis or degradation products of the chemicalcomposition. The hydrolysis or degradation products may optionally bemeasured following either partial purification or isolation by any knownseparation method, as described previously.

[0097] Mass spectrometers convert components of a sample into rapidlymoving gaseous ions and separate them on the basis of theirmass-to-charge ratios. The distributions of isotopes or isotopologues ofions, or ion fragments, may thus be used to measure the isotopicenrichment in one or more chemical compositions, or chemical orbiochemical degradation products.

[0098] Generally, mass spectrometers comprise an ionization means and amass analyzer. A number of different types of mass analyzers are knownin the art. These include, but are not limited to, magnetic sectoranalyzers, electrostatic analyzers, quadrupoles, ion traps, time offlight mass analyzers, and fourier transform analyzers. In addition, twoor more mass analyzers may be coupled (MS/MS) first to separateprecursor ions, then to separate and measure gas phase fragment ions.

[0099] Mass spectrometers may also include a number of differentionization methods. These include, but are not limited to, gas phaseionization sources such as electron impact, chemical ionization, andfield ionization, as well as desorption sources, such as fielddesorption, fast atom bombardment, matrix assisted laserdesorption/ionization, and surface enhanced laser desorption/ionization.

[0100] In addition, mass spectrometers may be coupled to separationmeans such as gas chromatography (GC) and high performance liquidchromatography (HPLC). In gas-chromatography mass-spectrometry (GC/MS),capillary columns from a gas chromatograph are coupled directly to themass spectrometer, optionally using a jet separator. In such anapplication, the gas chromatography (GC) column separates samplecomponents from the sample gas mixture and the separated components areionized and chemically analyzed in the mass spectrometer.

[0101] Various mass spectrometers and combinations of separationtechnologies and mass spectrometers are contemplated for use in theinvention including, but not limited to, gas chromatography/massspectrometry, liquid chromatography-mass spectrometry, gaschromatography-Pyrolysis-isotope ratio/mass spectrometry, or gaschromatography-combustion-isotope ratio/mass spectrometry, cycloidalmass spectrometry, Fourier-transform-isotope ratio (IR)-spectroscopy,near IR laser spectroscopy, or isotope ratio mass spectrometry

[0102] b. Metabolism

[0103] Very low quantities of labeled water may be detected. In oneembodiment, 1 part in 10³ labeled water may be identified. In anotherembodiment, 1 part in 10⁴ labeled water may be identified. In anotherembodiment, 1 part in 10⁵ labeled water may be identified. In anotherembodiment, 1 part in 10⁶ labeled water may be identified. In anotherembodiment, 1 part in 10⁷ labeled water may be identified.

[0104] 1. Detecting Water Following Sugar Metabolism

[0105] The methods of measuring the consequences of sugar ingestion maybe accomplished by measuring sugar metabolism products. The rate ofmetabolic water production from the oxidation of fuels, includingsugars, is sufficient to achieve relatively high levels of labeled waterwhen modest doses of compounds containing labeled sugars areadministered.

[0106] Alternatively, labeled glucose may be polymerized to formlabeled, glycogen, which may then be measured.

[0107] 2. Detecting Water Following Fatty Acid Metabolism

[0108] The methods of measuring the consequences of fatty acid ingestionmay be accomplished by measuring fatty acid metabolism products. Therate of metabolic water production from fatty acid oxidation(metabolism) is sufficient to achieve relatively high levels of labeledwater, particularly ²H₂O, when modest doses of labeled fatty acids orcompounds containing fatty acid residues are administered.

[0109]FIG. 3 depicts the fatty acid metabolism pathway using deuterium²H-labeled fatty acids. Fatty acids ingested by an individual aredelivered to tissues, optionally stored as triacyl-glycerol, orconverted to water by β-oxidation. Labeled water may then be returned tothe blood stream, and incorporated into bodily fluids.

[0110] Labeled water may then be detected to determine the degree oflabel incorporation.

[0111] iv) The Additional Step of Measuring ²H Incorporated Into One orMore Chemical Compositions

[0112] The invention also contemplates the additional step of measuring²H incorporated into one or more chemical compositions in addition towater. Incorporation of labeled water generated from either labeledglucose or labeled fatty acid metabolism, can be used to measure othersynthesis and storage pathways in an organism (FIGS. 1 and 2). Thesepathways include protein synthesis, lipid synthesis (triglyceridesynthesis and cholesterogenesis), new fat synthesis (de novolipogenesis), and DNA synthesis (cell proliferation). The addition ofthese supplemental measurements (FIG. 3) adds further information to the²H-fatty acid or ²H-glucose labeling strategies.

[0113] One or more chemical compositions may be obtained, and optionallypartially purified or isolated, from the biological sample usingstandard biochemical methods known in the art. Chemical compositionsinclude, but are not limited to, glucose, glycogen,glycerol-triglyceride, triglyceride fatty acid, proteins, and DNA.Optionally, fragments of the compositions may also be obtained. Thefrequency of biological sampling can vary depending on differentfactors. Such factors include, but are not limited to, the nature of thechemical composition tested, ease of sampling, and half-life of a drugused in a treatment if monitoring responses to treatment.

[0114] In one variation, the one or more chemical compositions may beglucose. In a further variation, the dilution of orally administeredlabeled sugars, particularly ²H-glucose, in plasma glucose load revealsendogenous glucose production (EGP, FIG. 3). Considerable informationcan be gained about glucose utilization and synthesis pathways in thebody by use of this approach. FIG. 3 depicts the glucose metabolismpathway, specifically for deuterium labeled glucose. Glucose ingested byan individual is delivered to tissues, optionally stored as glycogen, orconverted to water and carbon dioxide via glycolysis and the citric acidcycle. Labeled water, particularly ²H₂O, may then be returned to theblood stream, and incorporated into bodily fluids, then intobiosynthetic products. In a still further variation, the proportion ofglucose may be used to identify the proportion of administered²H-labeled glucose undergoing glycolysis.

[0115] In another variation, the method may be used to determine newlysynthesized glycogen. Newly synthesized glycogen can be determinedindirectly by subtracting glycolysis from the total amount of glucoseinitially administered since the total disappearance of glucose is equalto the total amount of glycolysis+the total amount of newly synthesizedglycogen (FIGS. 2 and 3). The following equation can be used tocalculate newly synthesized glycogen:

Total glucose−glycolysis=newly synthesized glycogen

[0116] Determining newly synthesized glycogen is useful because it iswidely believed to be an early marker for an elevated risk of insulinresistance, diabetes and cardiovascular disease. That is, the moreglycogen formed from a given amount of glucose administered (as opposedto more glycolysis and less glycogen formed) the higher the risk fordeveloping insulin resistance, diabetes and cardiovascular disease.Glycogen formation is also an early marker for an elevated risk ofcerebrovascular disease.

[0117] In another variation, the rate of appearance of glycogen(Ra_(glycogen)), i.e., the rate of glycogen synthesis, may be determinedby subtracting the rate of appearance of glycolysis (Ra_(glycolysis))from the rate of disappearance of glucose (Rd_(glucose)). The followingequation can be used to calculate the rate of new glycogen synthesis:

Rd _(glucose) —Ra _(glycolysis) =Ra _(glycogen)

[0118] Knowing the rate of appearance of glycogen (i.e., the rate ofglycogen synthesis) provides additional useful information as a slowerrate of glycogen synthesis may be associated with an elevated risk ofdeveloping diabetes, cardiovascular disease, and cerebrovasculardisease. Furthermore, a slower rate of glycogen synthesis together withan increase in newly synthesized glycogen may be associated with anelevated risk of developing diabetes, cardiovascular disease, andcerebrovascular disease.

[0119] Similarly, knowing glycolysis may also be useful for determiningan elevated risk of diabetes, cardiovascular disease, andcerebrovascular disease. Knowing the rate of glycolysis providesadditional information useful for determining an elevated risk, asdescribed above for the rate of glycogen synthesis. Glycolysis and therate of glycolysis may be determined by measuring the amount of ²H₂Oformed after administration of ²H-Glucose, as is described supra.

[0120] In another variation, the one or more chemical compositions maybe glycogen. Glycogen may be measured directly by direct sampling usinginvasive or non-invasive procedures well known in the art. In a furthervariation, the one or more chemical compositions may be triglycerides.In a further variation, the method may be used to determine newtriglyceride synthesis.

[0121] In another variation, the one or more chemical compositions maybe compounds that include triglyceride-fatty acids. In a furthervariation, the method may be used to calculate new fatty acid synthesis.

[0122] In a still further variation, the method may be used to calculatethe ratio of labeled fatty acids to stored fatty acids. In a stillfurther variation, the method may be used to calculate the proportion ofadministered fatty acids undergoing fatty acid oxidation.

[0123] In another variation, the chemical composition may include aprotein.

[0124] In a further variation, the composition may include DNA. Themeasurement of DNA incorporation may then be used to determine the rateof new cell proliferation.

[0125] The one or more chemical compositions may also be purified,partially purified, or optionally, isolated, by conventionalpurification methods including high performance liquid chromatography(HPLC), fast performance liquid chromatography (FPLC), gaschromatography, gel electrophoresis, and/or any other separationmethods.

[0126] The one or more chemical compositions may be hydrolyzed orotherwise degraded to form smaller subunits. Hydrolysis or otherdegradation methods include any method known in the art, including, butnot limited to, chemical hydrolysis (such as acid hydrolysis) andbiochemical hydrolysis (such as enzyme cleavage or degradation).Hydrolysis or degradation may be conducted either before or afterpurifying and/or isolating the one or more chemical compositions. Forexample, polymers formed of monosaccharides may be degraded to formsmaller units of multiple monosaccharide residues, and/or optionally,monosaccharide constituents. Glycogen may be degraded chemically orproteolytically to form polysaccharides formed from glucose residues, oroptionally, glucose monomers. Proteins may be chemically orproteolytically degraded to form oligopeptides, or optionally, aminoacids. Fatty acids may be degraded to form ketone bodies, carbondioxide, and water. DNA may be degraded to form polynucleotides,oligonucloetides, nucleotides, nucleosides, nucleic acid bases, ornucleic acid backbones. Degradation products may be partially purified,or optionally, isolated, by conventional purification methods includinghigh performance liquid chromatography (HPLC), fast performance liquidchromatography (FPLC), gas chromatography, gel electrophoresis, and/orany other methods known in the art.

[0127] v) Calculating Kinetic Parameters

[0128] Rates or total amounts of ²H incorporation into water may becalculated. Rates of incorporation into other biopolymers may also becalculated. In one variation, the rate of incorporation of ²H into watermay be calculated. In another variation, the rate of degradation ofcompounds containing labeled sugars or fatty acids may be measured. In afurther variation, the biosynthesis and degradation rates of biopolymerssuch as glucose, glycogen, glycerol-triglyceride, triglyceride fattyacid, proteins, and DNA may also be determined. In still anothervariation, both rates of labeled water formation and biopolymerformation may be calculated. Finally, the rates may be usedindividually, or in combination, to diagnose, prognose, or identify therisk of metabolic or metabolically-related diseases or disorders.

[0129] Synthesis and degradation rates may be calculated by massisotopomer distribution analysis (MIDA), which may be used to calculatethe degradation or biosynthesis rates of metabolites and/or water bymeasuring the production of labeled water. In addition, MIDA may be usedto calculate the synthesis rate of biopolymers such as glucose,glycogen, glycerol-triglyceride, triglyceride fatty acid, proteins, andDNA after the sugar or fatty acid containing metabolites aremetabolized.

[0130] Variations of the MIDA combinatorial algorithm are discussed in anumber of different sources known to one skilled in the art.Specifically, the MIDA calculation methods are the subject of U.S. Pat.No. 5,336,686, incorporated herein by reference. The method is furtherdiscussed by Hellerstein and Neese (1999), incorporated herein byreference, as well as Patterson and Wolfe (1993), and Kelleher andMasterson (1992).

[0131] In addition to the above-cited references, calculation softwareimplementing the method is publicly available from Marc Hellerstein atthe University of California, Berkeley.

[0132] In brief, calculation of the number (n) of metabolicallyexchanged H-atoms between sugars or fatty acids and cellular water wasby combinatorial analysis, or MIDA. The relative fraction ofdouble-labeled to single-labeled sugars or fatty acid molecules revealsn if the precursor pool enrichment of ²H (p) is known. If one assumesthat p reflects body labeled water enrichment, then n can be calculatedby combinatorial analysis.

[0133] Fractional abundances of mass isotopomers result from mixingnatural abundance molecules with molecules newly synthesized from a poolof labeled monomers characterized by the parameters. A mixture of thistype can be fully characterized by f, the fraction new, and p. Thealgorithm proceeds in step-wise fashion, beginning with the simplestcalculation, a molecule synthesized from a single element containingisotopes with the same fractional abundances that occur in nature andnot mixed with any other molecules. We then proceed to moleculescontaining more than one element with all isotopes at natural abundance;then to non-polymeric molecules containing different elements, some ofwhich are in groups whose isotope composition is not restricted tonatural abundance but is variable; then to polymeric moleculescontaining combinations of repeating chemical units (monomers), whereinthe monomers are either unlabeled (containing a natural abundancedistribution of isotopes) or potentially labeled (containing anisotopically-perturbed element group); and finally to mixtures ofpolymeric molecules, composed of both natural abundance polymers andpotentially labeled polymers, the latter containing combinations ofnatural abundance and isotopically-perturbed units.

[0134] The last-named calculation addresses the condition generallypresent in a biological system, wherein polymers newly synthesizedduring the period of an isotope incorporation experiment are presentalong with pre-existing, natural abundance polymers and the investigatoris interested in determining the proportion of each that is present, inorder to infer synthesis rates or related parameters.

Methods of Use

[0135] Using the methods disclosed herein, metabolic consequences ofnutrient ingestion may be determined for a number of metabolites in anindividual. These consequences may be applied for diagnostic and/ormonitoring uses. There are numerous research and clinical applicationsof this technique.

[0136] In one variation, the effect of a drug agent on an individual maybe monitored. A change in the sugar or fatty acid metabolism in anindividual to which a drug agent has been administered identifies thedrug agent as capable of altering the sugar or fatty acid metabolism ofthe individual. The drug agent may be administered to the sameindividual, or different living systems. Drug agents may be any chemicalcompound or composition known in the art. Drug agents include, but arenot limited to, any chemical compound or composition disclosed in, forexample, the 13th Edition of The Merck Index (a U.S. publication,Whitehouse Station, N.J., USA), incorporated herein by reference in itsentirety.

[0137] In another variation, drug agents can be at least partiallyidentified as to desirable or undesirable (or both) characteristics.Such information is useful in evaluating whether a drug agent should beadvanced in clinical development, for example, whether a drug agentshould be tested in in vivo animal models, whether it should be thesubject of clinical trials, and whether it should be advanced further inthe clinical trial setting (e.g., after an IND filing and/or aftercompletion of phase I, phase II and/or phase III trials). Once advancedthrough the filing and approval of an NDA, it is readily apparent thatthe methods of the present invention allow for the early identificationof drug agents useful in the treatment of metabolic diseases such asdiabetes, cardiovascular disease, and other obesity-related diseases ordisorders. In another embodiment, the fate of nutrients as surrogatesduring FDA trials may be monitored.

[0138] In another variation, the methods may be used to identifyindividuals at risk for diabetes. In another variation, the methods maybe used to identify patients at risk for high-fat diet-induced obesity.

[0139] In another variation, the methods may be used to diagnose,prognose, or identify the risk of insulin resistance/diabetes mellitus(type II diabetes) in an individual. In a further variation, the methodsmay be used to diagnose, prognose, or identify the risk of high-fatdiet-induced obesity in an individual. In another variation, the methodsmay be used to monitor the effects of interventions or treatment methodsto prevent or reverse insulin resistance/diabetes mellitus or high-fatdiet-induced obesity.

Isotopically-perturbed Molecules

[0140] In another variation, the methods provide for the production ofisotopically-perturbed molecules (e.g., labeled fatty acids, lipids,carbohydrates, proteins, nucleic acids and the like). Theseisotopically-perturbed molecules comprise information useful indetermining the flux of molecules within the metabolic pathways ofinterest. Once isolated from a cell and/or a tissue of an organism, oneor more isotopically-perturbed molecules are analyzed to extractinformation as described, supra.

[0141] In other variations, the methods may be used to diagnose or treatwasting diseases or disorders, hypoglycemia, or glycogen storagedisease.

Kits

[0142] In another aspect, the invention provides kits for analyzing themetabolic fate of glucose or fatty acids in vivo. The kits may includelabeled glucose or fatty acids. The kits may also include chemicalcompounds known in the art for isolating chemical and biochemicalcompounds from urine, bone, or muscle and/or chemicals necessary to geta tissue sample, automated calculation software for combinatorialanalysis, and instructions for use of the kit are optionally included inthe kit.

[0143] Other kit components, such as tools for administration ofcompounds containing labeled sugars and fatty acids are optionallyincluded. Tools may include measuring cups, needles, syringes, pipettes,IV tubing), may optionally be provided in the kit. Similarly,instruments for obtaining samples from the subject (e.g., specimen cups,needles, syringes, and tissue sampling devices) may also be optionallyprovided.

Information Storage Devices

[0144] The invention also provides for information storage devices suchas paper reports or data storage devices comprising data collected fromthe methods of the present invention. An information storage deviceincludes, but is not limited to, written reports on paper or similartangible medium, written reports on plastic transparency sheets ormicrofiche, and data stored on optical or magnetic media (e.g., compactdiscs, digital video discs, magnetic discs, and the like), or computersstoring the information whether temporarily or permanently. The data maybe at least partially contained within a computer and may be in the formof an electronic mail message or attached to an electronic mail messageas a separate electronic file. The data within the information storagedevices may be “raw” (i.e., collected but unanalyzed), partiallyanalyzed, or completely analyzed. Data analysis may be by way ofcomputer or some other automated device or may be done manually. Theinformation storage device may be used to download the data onto aseparate data storage system (e.g., computer, hand-held computer, andthe like) for further analysis or for display or both. Alternatively,the data within the information storage device may be printed ontopaper, plastic transparency sheets, or other similar tangible medium forfurther analysis or for display or both. The information storage devicemay provide for retrieval of the data. Such retrieval can be for thepurpose of display and/or for further analysis or for any other purpose.

[0145] The following examples are provided to show that the methods ofthe invention may be used to determine the fate of metabolic glucose orfatty acids. Those skilled in the art will recognize that while specificembodiments have been illustrated and described, they are not intendedto limit the invention.

EXAMPLES Example 1 Kinetic OGTT—Glycolytic Disposal of Glucose in NormalRats and Mice

[0146] The kinetic oral glucose tolerance test for mice and rats isdepicted in FIGS. 5 and 6, respectively. The figures depict percentglycolysis, measured by deuterium incorporation into water followingadministration of deuterium-labeled glucose.

[0147] Sprague-Dawley rats (200-250 g, Simonsen Inc., Gilroy Calif.) andC57Blk/6ksj mice (10-15 g, Jackson Laboratories, Bar Harbor Me.) wereused. Housing was in individual cages for rats and groups of 5 for mice.Feeding was ad-libitum with Purina® rodent chow. All studies receivedprior approval from the UC Berkeley Animal Care and Use Committee.The²H-glucose labeling protocol consisted of an initial intraperitoneal(ip) injection of 99.9% [6,6-²H₂] glucose. For labeling rats and mice, 2mg labeled glucose per gram body weight were introduced. Body water wascollected as serum at various timepoints.

[0148] Glycolysis was measured by measuring deuterium in body water as apercent of administered [6,6-²H₂] glucose normalized to account fordifferent molar quantities of deuterium in molecular glucose andmolecular water. Deuterized water was measured by isotope ratio massspectrometry.

Example 2 Kinetic OGTT—Glycolytic Disposal of Glucose in Normal Rats andMice

[0149] A kinetic oral glucose tolerance test in a human subject isdepicted in FIG. 4. The figure depicts percent glycolysis, measured bydeuterium incorporation into water following ingestion of deuteriumlabeled glucose.

[0150] The ²H-glucose labeling protocol consisted of an oral ingestionof 99.9% [6,6-²H₂] glucose. 15 grams glucose in 50 grams oral load (30%[6,6-²H₂]) were ingested by the human subject. Body water was collectedas serum at various timepoints.

[0151] Glycolysis was measured by measuring deuterium in body water as apercent of administered [6,6-²H₂] glucose normalized to account fordifferent molar quantities of deuterium in molecular glucose andmolecular water. Deuterized water was measured by isotope ratio massspectrometry.

Example 3

[0152] [6,6-²H₂] glucose was administered orally (15 grams in water) toa lean male human subject (Subject #1), to an overweight but not obesemale human subject (Subject #2), to an obese female human subject(Subject #3), and to a lean male human subject with HIV/AIDS (Subject#4). Blood samples were collected (10 cc) every hour for four hours. ²Hcontent of blood glucose was measured by isolating glucose from bloodand preparing into a form compatible with isotope ratio massspectrometry. The isotopic (²H₂O) content of body water was measured byisolating water from the blood and preparing into a form compatible withisotope ratio mass spectrometry. Mass spectrometry was performed tocalculate the fraction of ²H from ²H-glucose released into body water.This represents glycolysis/oxidation from the administered glucose load.Measurement of ²H-glucose content measured by mass spectrometry wascompared to administered ²H content of administered ²H-glucose tocalculate the body's production rate of glucose. Fasting plasma insulinlevels were measured by radioimmunoassay (RIA) specific for insulin. RIAkits are readily available from a variety of commercial sources such asLinco Research Inc., St. Charles, Mo., USA or Phoenix Pharmaceutical,Inc., Belmont, Calif., USA. Plasma glucose levels were measured by theuse of glucose oxidase, a technique well known in the art. Kitscontaining glucose oxidase for the measurement of glucose are readilyavailable, for example from Sigma Aldrich, St. Louis, Mo., USA. Table 1depicts the results. TABLE 1 Subjects Undergoing ²H-OGTT (75 g glucose;15 g [6,6-²H] Glucose) Fasting Glycolysis Plasma Peak Plasma (mMoles²H₂OSubject BMI Insulin Glucose produced) # (kg/m²) Gender (μU/mL) (mg/dL) 2h 4 h 1 23.5 M <15 105 25 50 2 27.2 M 20 96 26 41 3 31.8 F 33 108 15 344 23.5 M >30 225 16 33

[0153] As can be seen from table 1, supra, Subject #1 is a normal, leanhealthy male subject (normal control). Subject #2 is a normal,overweight but not obese healthy male subject. Subject #3 is a normal,obese healthy female. Subject #4 is a lean male with HIV/AIDS. The dataidentifies clinically evident glucose intolerant or diabetic individualsdespite the absence of obesity (Subject #4). Subject #4 is an HIVpositive male with AIDS receiving protease-inhibitor containinganti-retroviral therapy. People with AIDS who receive anti-retroviraltreatments often develop a glucose intolerant or diabetic phenotype. Thedata in table 1 also identifies pre-diabetic (insulin resistant)individuals before glucose intolerance is apparent (Subject #3). Thismethod allows for early detection of glucose intolerance.

References

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[0166] 13. Hellerstein M K, Neese R A, Kim Y-K, Schade-Serin V, CollinsM. Measurement of synthesis rates of slow-turnover proteins from ²H₂Oincorporation into non-essential amino acids (NEAA) and application ofmass isotopomer distribution analysis (MIDA). FASEB J 16:A256, 2002.

[0167] 14. Kim Y-K, Neese R A, Schade-Serin V, Collins M, Misell L,Hellerstein M K. Measurement of synthesis rates of slow-turnoverproteins based on ²H₂O incorporation into non-essential amino acids andapplication of mass isotopomer distribution analysis. Biochemical J,Submitted, 2002.

[0168] 15. Reaven G M. Banting lecture 1988. Role of insulin resistancein human disease. Diabetes 37(12):1595-607, 1988.

[0169] All publications, patents, and patent applications cited hereinare hereby incorporated by reference in their entirety for all purposesto the same extent as if each individual publication, patent, or patentapplication were specifically and individually indicated to be soincorporated by reference. Although the foregoing invention has beendescribed in some detail by way of illustration and example for purposesof clarity of understanding, it is readily apparent to those of ordinaryskill in the art in light of the teachings of this invention thatcertain changes and modifications may be made thereto without departingfrom the spirit and scope of the claims.

[0170] Applicants have not abandoned or dedicated to the public anyunclaimed subject matter.

I claim:
 1. A method of determining the metabolism of one or more sugarsor fatty acids in an individual, said method comprising: (a)administering one or more compositions comprising one or more ²H-labeledsugars or ²H-labeled fatty acids to an individual; (b) obtaining one ormore bodily tissues or fluids at one or more times from said individual;and (c) detecting the incorporation of said ²H from said one or more²H-labeled sugars or ²H-labeled fatty acids into water to determine themetabolism of said one or more sugars or fatty acids in said individual.2. The method according to claim 1, wherein said one or morecompositions comprise ²H-labeled glucose.
 3. The method according toclaim 2, wherein said ²H-labeled glucose is chosen from[6,6-²H₂]glucose, [1-²H₁]glucose, and [1,2,3,4,5,6-²H₇]glucose.
 4. Themethod according to claim 1, wherein said one or more compositions areadministered by a technique chosen from oral, gavage, intraperitoneal,intravascular, and subcutaneous administration.
 5. The method accordingto claim 4, wherein said one or more compositions are administeredorally.
 6. The method according to claim 1, wherein said individual is amammal.
 7. The method according to claim 6, wherein said mammal ischosen from humans, rodents, primates, hamsters, guinea pigs, dogs, andpigs.
 8. The method according to claim 7, wherein said mammal is ahuman.
 9. The method according to claim 1, wherein said one or morebodily tissues or fluids are chosen from blood, urine, saliva, andtears.
 10. The method of claim 1, wherein said one or more bodilytissues or fluids are chosen from liver, muscle, adipose, intestine,brain, and pancreas.
 11. The method of claim 1, comprising theadditional step of partially purifying said water.
 12. The method ofclaim 11, comprising the additional step of isolating said water. 13.The method according to claim 1, comprising the additional step ofmeasuring ²H incorporation or incorporation ratio into one or morechemical compositions chosen from glucose, glycogen,glycerol-triglyceride, triglyceride fatty acid, proteins, and DNA. 14.The method according to claim 13, wherein said chemical composition isglucose.
 15. The method according to claim 14, comprising the additionalstep of measuring endogenous glucose production.
 16. The methodaccording to claim 14, comprising the additional step of measuring theproportion of labeled glucose stored in tissue glycogen relative to saidlabeled sugar administered.
 17. The method according to claim 14,comprising the additional step of measuring the proportion or rate ofadministered ²H-glucose undergoing glycolysis.
 18. The method accordingto claim 13, wherein said chemical composition is glycogen.
 19. Themethod according to claim 13, wherein said chemical composition isglycerol-triglyceride.
 20. The method according to claim 19, comprisingthe additional step of calculating new triglyceride synthesis.
 21. Themethod according to claim 13, wherein said chemical composition istriglyceride fatty acid.
 22. The method according to claim 21,comprising the additional step of calculating new fatty acid synthesis.23. The method according to claim 13, comprising the additional step ofcalculating the proportion or storage rate of labeled fatty acids storedin tissue relative to labeled fatty acid administered.
 24. The methodaccording to claim 1, comprising the additional step of calculating theproportion or storage rate of administered labeled fatty acidsundergoing fatty acid oxidation.
 25. The method according to claim 13,wherein said chemical composition is a protein.
 26. The method accordingto claim 13, wherein said chemical composition is DNA.
 27. The methodaccording to claim 24, comprising the additional step of calculating therate or amount of DNA synthesis.
 28. The method according to claim 1,further comprising calculating the rate or total amount of incorporationof said ²H into said water.
 29. The method according to claim 13,further comprising calculating the rate of or amount incorporation of ²Hinto said one or more chemical compositions.
 30. The method according toclaim 28, further comprising calculating the rate of or amountincorporation of ²H into said one or more chemical compositions.
 31. Themethod according to claim 1, wherein said water is detected by methodschosen from gas chromatography/mass spectrometry, liquidchromatography-mass spectrometry, gas chromatography-pyrolysis-isotoperatio/mass spectrometry, gas chromatography-combustion-isotoperatio/mass spectrometry, cycloidal mass spectrometry,Fourier-transform-isotope ratio (IR)-spectroscopy, near IR laserspectroscopy, and isotope ratio mass spectrometry.
 32. The methodaccording to claim 1, wherein said detecting step may be accomplished bydetecting one part ²H in 10⁷ parts water.
 33. A method of identifyingthe effect of a drug agent on an individual, comprising: administering adrug agent to the individual; determining the metabolism of one or moresugars or fatty acids in the individual according to claim 1 to identifythe effect of said drug agent on said individual.
 34. The methodaccording to claim 1, wherein said metabolism determination is used as asurrogate marker for FDA approval of drugs.
 35. The method according toclaim 1, wherein said metabolism determination is used for the clinicalmanagement of patients.
 36. A method of diagnosing insulin resistance ordiabetes mellitus, comprising: determining the metabolism of one or moresugars or fatty acids according to claim
 1. 37. The method according toclaim 1, wherein said metabolism determination is chosen fromidentifying individuals at risk for insulin resistance and diabetesmellitus.
 38. The method according to claim 1, wherein said metabolismdetermination further comprises diagnosing high-fat diet-inducedobesity.
 39. The method according to claim 1, wherein said metabolismdetermination further comprises identifying individuals at risk forhigh-fat diet-induced obesity.
 40. The method according to claim 1,wherein said metabolism determination further comprises the step chosenfrom monitoring the effects of interventions to prevent or reverseinsulin resistance, diabetes mellitus and high-fat diet-induced obesity.41. The method according to claim 1, comprising the further step chosenfrom diagnosing and treating wasting disorders.
 42. The method accordingto claim 1, comprising the further step chosen from diagnosing andtreating hypoglycemia.
 43. The method according to claim 1, comprisingthe further step chosen from diagnosing and treating glycogen storagedisease.
 44. A kit for determining the metabolism of a sugar in anindividual comprising: a) one or more labeled sugars, b) instructionsfor use of the kit, wherein the kit is used to determine sugarmetabolism in said individual.
 45. The kit of claim 44 furthercomprising chemical compounds for isolating water.
 46. The kit of claim44 further comprising chemical compounds for isolating a compositionchosen from glucose, glycogen, proteins, and DNA.
 47. The kit of claim44 further comprising a tool for administering labeled glucose.
 48. Thekit of claim 44 further comprising an instrument for collecting a samplefrom the subject.
 49. A drug agent the effect of which was at leastpartially identified by the method of claim
 33. 50. An isotopicallyperturbed molecule chosen from glycogen, glycerol-triglyceride,triglyceride fatty acid, proteins, and DNA.
 51. The method according toclaim 33, further comprising the manufacturing of one or more drugagents at least partially identified by said method.
 52. An informationstorage device comprising data obtained from the method according toclaim
 1. 53. The device of claim 52, wherein said device is a printedreport.
 54. The printed report of claim 53, wherein the medium in whichsaid report is printed on is chosen from paper, plastic, and microfiche.55. The device of claim 52, wherein said device is a computer disc. 56.The disc of claim 55, wherein said disc is chosen from a compact disc, adigital video disc, and a magnetic disc.
 57. The device of claim 52,wherein said device is a computer.
 58. An information storage devicecomprising data obtained from the method according to claim
 33. 59. Anisotopically-perturbed molecule produced by the method according toclaim
 1. 60. A kit for determining the metabolism of a fatty acid in anindividual comprising: a) one or more labeled fatty acids, and b)instructions for use of the kit, wherein the kit is used to determinefatty acid metabolism in said individual.
 61. The kit of claim 44further comprising chemical compounds for isolating water.
 62. The kitof claim 44 further comprising chemical compounds for isolating acomposition chosen from glycerol-triglyceride, triglyceride-fatty acid,proteins, and DNA.
 63. The kit of claim 44 further comprising a tool foradministering labeled fatty acids.
 64. The kit of claim 44 furthercomprising an instrument for collecting a sample from the subject. 65.At least one isolated deuterated water molecule (²H₂O), produced by themethod according to claim
 1. 66. At least one isolated deuterated watermolecule (²H₂O), produced by the method according to claim 33.